1. Field of the Invention
This invention relates to fragments of the Factor VIII procoagulant protein (Factor VIIIC), their recombinant DNA-directed synthesis, and their potential use in the treatment of patients who have developed antibodies which inhibit Factor VIII.
2. Brief Description of the Prior Art
Normal human plasma contains a complex of two proteins which is referred to as the Factor VIII complex. One component of the Factor VIII complex has antihemophilic factor procoagulant activity and is designated Factor VIIIC. A deficiency in Factor VIIIC is characteristic of hemophilia A, a disease transmitted by X-chromosomal inheritance.
The conventional treatment for hemophilia A is administration of Factor VIIIC (antihemophilic factor, or "AHF"), concentrated in any of various means from the plasma of donors. Some hemophiliacs exhibit, in effect, "resistance" to this treatment, in that administration of Factor VIIIC in doses which are usually effective for most hemophiliacs produces reduced or no therapeutic effect.
This phenomenon has generally been considered to be due to one or more antibody inhibitors of Factor VIIIC in the circulatory system of the afflicted individual. (Persons who are not hemophiliacs can also develop these inhibitors.) It may be possible to treat Factor VIIIC inhibitors by administering greatly increased amounts of Factor VIIIC, some of which serves to saturate the inhibitory capacity of the Factor VIIIC inhibitor and the remainder of which provides the desired therapeutic effect uninhibited However, this approach is extremely expensive, consumes large amounts of AHF per patient, and carries the risk of increasing the amounts of other products or agents, unavoidably present with the Factor VIIIC, to which the patient is exposed. Thus, there is a need for a product which effectively neutralizes the activity of Factor VIII inhibitors, without introducing unnecessary co-products.
In the bloodstream, the protease thrombin cleaves Factor VIIIC to yield fragments which are active in blood coagulation, and then it further cleaves some fragments into inactive species. These useful properties of thrombin can be employed in vitro to generate a series of unique subfragments of Factor VIIIC.
Recently, it has been shown by Fulcher et al., Proc. Natl. Acad. Sci. USA, 82:7728 (1985) that the patient inhibitor antibodies bind to only some of the Factor VIIIC fragments generated by thrombin cleavage, i.e., fragments of 44,000 d and 72,000 d. One option for removing inhibitor activity is thus to inject inhibitor patients only with the required Factor VIIIC fragments rather than the full-length molecule.
Genes coding for polypeptides such as Factor VIIIC or fragments of Factor VIIIC may be cloned by incorporating a DNA fragment encoding the polypeptide into a recombinant DNA vehicle (e.g., prokaryotic or eukaryotic vectors) and transforming a suitable host. Such recombinant DNA techniques have now become well known and are described in Methods In Enzymology, (Academic Press), Volumes 65 and 68 (1979), 100 and 101 (1983), and the references cited therein, which are incorporated herein by reference. An extensive technical discussion embodying most commonly used recombinant DNA methodologies can be found in Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982).
One way of obtaining a DNA fragment encoding a desired polypeptide such as Factor VIIIC is via cDNA cloning. In this process, messenger RNA (mRNA) is isolated from cells known or suspected of producing the desired protein. Through a series of enzymatic reactions, the mRNA population of the cells is copied into a complementary DNA (cDNA). The resulting cDNA is then inserted into cloning vehicles and subsequently used to transform a suitable prokaryotic or eukaryotic host. The resultant gene "library" is comprised of a population of transformed host cells, each of which contain a single gene or gene fragment. The entire library, therefore, provides a representative sample of the coding information present in the mRNA mixture used as a starting material.
Gene libraries are screened using specific nucleic acid or antibody probes. Nucleic acid probes are useful for locating cDNAs by hybridization and autoradiography techniques. This approach, however, requires previous knowledge of at least a portion of the protein's amino acid or DNA-encoding sequence. Alternatively, methods have been developed to identify specific clones by probing recombinant gene libraries with antibodies specific for the encoded protein of interest. This method can only be used with "expression vector" cloning vehicles since elaboration of the product protein is required. An example of this is the bacteriophage lambda gtll system described by Young and Davis, Proc. Natl. Acad. Sci. USA, 80:1194-1198 (1983) and Young and Davis, Science, 222:778 (1983).
Once isolated, these cDNAs can be genetically engineered. Gene fragments can be assembled into complete genes. Alternatively, as described in this invention, specific fragments of a gene can be engineered independently of the rest of the gene. Protein fragments encoded by these engineered gene fragments may not be found in nature, yet they may have significant utility in treating undesirable physiological conditions. The engineering of recombinant Factor VIIIC fragments for treatment of anti-Factor VIIIC antibody producing hemophiliac or non-hemophiliac patients is one such case.